Lesson 15

Question 1

in sickle cell disease, what type of mutation causes disease?

Answer 1

a single point mutation

Question 2

what specific point mutation causes sickle cell disease?

Answer 2

the substitution of glutamic acid with a valine in position 6, giving rise to a mutant global chain called HbS (sickle)

Question 3

what does HbS cause in erythrocytes?

Answer 3

in deoxygenation where HbS forms fibers (different from normal Hb which is present as globular) in a process called polymerization → these fibers are toxic inside erythrocytes thus damaging them, causing them to lose cations and water forming the sickle shape

Question 4

what are the major symptoms of sickle cell disease?

Answer 4

hemolytic anemia, vaso-occlusion, and morbidity and mortality

Question 5

what is vaso occlusion?

Answer 5

impaired rheology, adhesion between sickled erythrocytes, neutrophils, endothelium, and platelets

Question 6

what is hemolysis?

Answer 6

results in the release of the heme group, which is toxic and induces endothelial dysfunction (particularly toxic in the small vessels because damages the vessel walls, the endothelium), the release of cytokines, which results in an inflamed microenvironment, a sterile inflammation

Question 7

what other disease does the geographic distribution of sickle cell disease correlate with?

Answer 7

malaria

Question 8

what is the effect of hemolytic anemia?

Answer 8

blocks the small blood vessels

Question 9

what does vaso occlusion cause systemically?

Answer 9

causes ischemic damage to different tissues including the nervous system leading got stroke

Question 10

what are the chronic complications of sickle cell disease?

Answer 10

general vasculopathy and progressive ischemic organ damage in important organs like the kidneys, bones and liver

Question 11

currently what are the only two treatments for sickle cell disease?

Answer 11

bone marrow transplant or blood transfusion

Question 12

what does HPFH stand for?

Answer 12

hereditary persistence of fetal hemoglobin

Question 13

what is HPFH?

Answer 13

associated with high levels of fetal hemoglobin in adults (normally in adults we undergo a globin switch so we do not have any fetal hemoglobin)

Question 14

in what case is HPFH benign?

Answer 14

when the 𝛾-globin gene is reactivated

Question 15

what are the two molecular causes of HPFH?

Answer 15

point mutations in the HGB gene or locus (promotor of the 𝛾-globin) or deletions in the HGB gene or locus downstream of the 𝛾-globin gene

Question 16

what do point mutations in the HGB gene lead to?

Answer 16

non-deletional HPFH → in the γ-promoter we have binding sites for repressors: some mutations in here cause disruption of the repressor binding site ; there could also be the creation of an activator binding site

Question 17

what do deletions in the HGB gene cause?

Answer 17

lead to deletional HPFH → these kinds of mutations are not really fully explained - however, in some cases
there is a deletion of repressor binding sites, in some other cases it’s not clear why the deletion causes HPFH

Question 18

what can we induce for the reactivation of the 𝛾-globin gene?

Answer 18

induce NHEJ (non-homologous end joining) and disrupt the downstream gene, which is way easier → you can delete the gene for the repressor if you don’t want to have repression

Question 19

what three levels were identified to have high levels of HbF in HPHF patients?

Answer 19

1. In an intergenic region between HBS1L and MYB on chromosome 6
2. Inside the β-locus, some regions are responsible for reactivating γ-globin
3. A new candidate, which isthe BCL11A gene, located on chromosome 2

Question 20

what form of BCL11A has the ability to silence 𝛾 genes?

Answer 20

the erythroid form

Question 21

which other repressors form a complex with BCL11A to silence the 𝛾-genes?

Answer 21

LRF, GATA1, FOG1, and NuRD

Question 22

in what four ways can we induce HPFH?

Answer 22

1. target the BCL11A erythroid enhancer
2. induction of non-deletional HPFH
3. induction of deletional HPFH
4. gene repair

Question 23

how can we target the BCL11A erythroid enhancer?

Answer 23

we can delete the enhancer with zinc finger or Cas to obtain less BCL11A expression leading to the reactivation and expression of 𝛾 genes

Question 24

how can we induce non-deletional HPFH?

Answer 24

by creating mutations on the binding sites on the promotors of 𝛾-genes to not allow the binding of repressors

Question 25

how can we induce deletional HPFH?

Answer 25

using zinc finger, Cas9, or NHEJ to delete the repressors binding sites resulting in big deletions

Question 26

how can we use gene repair to induce HPFH?

Answer 26

we can induce the repair of the mutated gene through HDR (clinically speaking currently this is not really possible since HDR occurs at very low frequencies)

Question 27

how can HbF be therapeutic?

Answer 27

it has anti-sickling properties as HbF is able to repel HbS polymerization → the presence of HbF impedes the polymerization of HbS

Question 28

what is an important characteristic of HbF?

Answer 28

it is an anti-sickling molecule

Question 29

how can we use gene correction to treat β-thalassemia?

Answer 29

with gene correction we have low efficiency in hematopoietic stem cells (HSCs), meaning that a lot of work is still ongoing, because this will be a
very valid approach to replace the mutated genes, so not the aim is to optimize the reagents to achieve higher efficiency

Question 30

how was targeting used to treat β-thalassemia with gene correction?

Answer 30

a specific place in the Hb locus was targeted (more than just a replacement)

Question 31

how was the Hb locus targetted, and what was this the first use of?

Answer 31

the first use of ribonuclear particle, instead of just mRNA, so in this case it was clear that the efficiency of indels (of
cutting) was much higher if I provided all the system using Rnp instead of just mRNA.

Question 32

describe enhancer disruption in BCL11A when treating β-thalassemia:

Answer 32

in this case we have a completely different realist because the efficiency of non-homologous end joining is very high

Question 33

how does enhancer disruption work?

Answer 33

• one is disrupting a the coding part of the gene, and the gene editing tool used in this case are the ZFNs homologous to the coding region
• another couple of ZFNs targets the enhancer sequence (so not the coding sequence, just in a regulatory element promoting the expression of BCL11A in the erythroid lineages)

Question 34

what is an important step to consider when working with ZFN or with any other gene editing strategies?

Answer 34

you must be aware of off targets → the
system has to be very specific in order to cut the human genome just in the desired region

Question 35

how do ZFNs effect the genome in enhancer disruption?

Answer 35

most of the genome editing sequence give a KO because most of them are deletion that make the sequence go out of frame

Question 36

if you eliminate the sequence. what occurs?

Answer 36

you will not have expression of BCL11A in al the cells in which it is expressed, while targeting the specific erythroid enhancer we delete it only from erythroid cells

Question 37

how is genome editing used to reproduce and generate the naturally occurring mutations associated with HPFH?

Answer 37

mutation in the γ promoter creates an E-box motif which composes a consensus sequence for an activator,
which is the TAL1 erythroid specific activator, created on the antisense strand of the γ-globin promoter

Question 37

how is genome editing used to reproduce and generate the naturally occurring mutations associated with HPFH?

Answer 37

mutation in the γ promoter creates an E-box motif which composes a consensus sequence for an activator,
which is the TAL1 erythroid specific activator, created on the antisense strand of the γ-globin promoter

Question 38

how is genome editing used to reproduce and generate the naturally occurring mutations associated with HPFH?

Answer 38

mutation in the γ promoter creates an E-box motif which composes a consensus sequence for an activator,
which is the TAL1 erythroid specific activator, created on the antisense strand of the γ-globin promoter

Question 39

what does the deletional HPFH mutation copy?

Answer 39

the 13 nucleotide deletion → done in HUDEP cell line

Question 40

out of all all the ways to induce HPFH, what is the most effective?

Answer 40

the elimination of the erythroid specific BCL11A expression

Question 41

what was the specific goal of both clinical trials to treat β-thalassemia and sickle cell disease?

Answer 41

to elevate HbF to achieve a therapeutic effect

Question 42

what occurred in the CTX001 clinical trial?

Answer 42

received autologous CD34+ cells but
cells were modified with Crispr-Cas9 and the guide targeting erythroid specific lineage enhancer BLC11A
(instead of lentiviral vectors)

Question 43

what was the CTX001 clinical trial the first to use?

Answer 43

genome editing with CRISPR-Cas9 ex vivo via transplantation into the patient

Question 44

what is the rational in the CTX001 study?

Answer 44

disrupt GATA1 binding site in the BCL11A promotor only present in the erythroid lineage

Question 45

what is different about gene editing in animal models compared to most other experiments?

Answer 45

genome editing works well in human cells but not so good in murine cells - one of the few cases where a technique works better in humans than in animal clinical trials

Question 46

for the β-thalassemia patients, what was the outcome of the clinical trial?

Answer 46

no more transfusions were needed in the 20 months of follow up → the level of adult Hb decreases, and HbF starts to appear and increases in quantity from the 2nd month to the 18th, while the level of transfused Hb keeps fading away because red cells die in the meantime

Question 47

what was the result of the CTZ001 clinical trial in SCD patients?

Answer 47

we can notice a big quantity of HbA because the patients are transfused a lot after the transplantation; after that, levels of HbF starts to grow up, even though we’re not eliminating sickling Hb, but just diluting it: the dilution has anti-sickling activity. That was enough in this patient, that indeed did not
experience any vaso-occlusive crisis in the follow up